Guro Stødle

Functional Toll-like receptors in primary first-trimester trophoblasts.

Toll-like receptors (TLRs) are an important part of the bodys danger response system and crucial for initiating inflammation in response to cellular stress, tissue damage, and infections. Proper placental development is sensitive to inflammatory activation, and a role for TLRs in trophoblast immune activation has been suggested, but no overall examination has been performed in primary trophoblasts of early pregnancy. This study aimed to broadly examine cell surface and endosomal TLR gene expression and activation in first-trimester trophoblasts. Gene expression of all ten TLRs was examined by quantitative RT-PCR (RT-qPCR) in primary first-trimester trophoblasts (n = 6) and the trophoblast cell line BeWo, and cytokine responses to TLR ligands were detected by quantitative multiplex immunoassay. Primary first-trimester trophoblasts broadly expressed all ten TLR mRNAs; TLR1, TLR2, TLR3, TLR4, and TLR6 mRNA were expressed by all primary trophoblast populations, while TLR5, TLR7, TLR8, TLR9, and TLR10 mRNA expression was more restricted. Functional response to ligand activation of cell surface TLR2/1, TLR4, and TLR5 increased IL-6 and/or IL-8 release (P < 0.01) from primary trophoblasts. For endosomal TLRs, TLR3 and TLR9 ligand exposure increased receptor-specific production of IL-8 (P < 0.01) and IFN-γ-induced protein 10 (IP-10; P < 0.001) or vascular endothelial growth factor A (VEGFA; P < 0.01). In contrast, BeWo cells expressed lower TLR mRNA levels and did not respond to TLR activation. In conclusion, primary first-trimester trophoblasts broadly express functional TLRs, with inter-individual variation, suggesting that trophoblast TLR2, TLR3, TLR4, TLR5, and TLR9 might play a role in early placental inflammation.

Toll-like receptor profiling of seven trophoblast cell lines warrants caution for translation to primary trophoblasts

INTRODUCTION Excessive placental inflammation is associated with pregnancy complications. Toll-like receptors (TLRs) are sensors for danger signals from infections and damaged tissue and initiate inflammation. Trophoblasts in the placenta broadly express TLRs. Trophoblast cell lines are used as surrogates for primary trophoblasts for in vitro studies, but the inflammatory translatability of trophoblast cell lines warrants examination. We aimed to assess TLR1-10 gene expression and activation in seven trophoblast cell lines and compare this to primary trophoblasts. METHODS The five choriocarcinoma trophoblast cell lines BeWo, JAR, JEG-3, AC1M-32 and ACH-3P, and the two SV40 transfected trophoblast cell lines HTR-8/SVneo and SGHPL-5 were included and compared to primary first trimester trophoblasts (n = 6). TLR1-10 gene expression was analyzed by RT-qPCR. Cells were stimulated by specific TLR1-9 ligands for 24 h and cytokine release was measured by a 10-plex immunoassay. RESULTS All choriocarcinoma cell lines demonstrated broad TLR gene expression, but lacked functional cytokine response to TLR ligand activation. In contrast, SV40 transfected cell lines showed restricted TLR gene expression, but SGHPL-5 cells displayed significantly increased levels of interleukin (IL)-6, IL-8, IL-12 and vascular endothelial growth factor A after TLR3 and/or TLR4 activation (P < 0.01), while TLR2 activation increased IL-6 and IL-8 levels (P < 0.05). HTR8/SVneo cells responded to TLR3 activation by increased IL-6 and interferon (IFN)-γ (P < 0.05). The SGHPL-5 TLR profile most closely resembled primary trophoblast. DISCUSSION The characterized trophoblast cell line TLR profiles serve as a reference and warrant caution when selecting trophoblast cell lines as in vitro models for immune responses in primary trophoblasts.

Placental inflammation in pre-eclampsia by Nod-like receptor protein (NLRP)3 inflammasome activation in trophoblasts: Placental inflammation by NLRP3

Pre‐eclampsia is associated with increased levels of cholesterol and uric acid and an inflamed placenta expressing danger‐sensing pattern recognition receptors (PRRs). Crystalline cholesterol and uric acid activate the PRR Nod‐like receptor protein (NLRP)3 inflammasome to release interleukin (IL)‐1β and result in vigorous inflammation. We aimed to characterize crystal‐induced NLRP3 activation in placental inflammation and examine its role in pre‐eclampsia. We confirmed that serum total cholesterol and uric acid were elevated in pre‐eclamptic compared to healthy pregnancies and correlated positively to high sensitivity C‐reactive protein (hsCRP) and the pre‐eclampsia marker soluble fms‐like tyrosine kinase‐1 (sFlt‐1). The NLRP3 inflammasome pathway components (NLRP3, caspase‐1, IL‐1β) and priming factors [complement component 5a (C5a) and terminal complement complex (TCC)] were co‐expressed by the syncytiotrophoblast layer which covers the placental surface and interacts with maternal blood. The expression of IL‐1β and TCC was increased significantly and C5a‐positive regions in the syncytiotrophoblast layer appeared more frequent in pre‐eclamptic compared to normal pregnancies. In‐vitro activation of placental explants and trophoblasts confirmed NLRP3 inflammasome pathway functionality by complement‐primed crystal‐induced release of IL‐1β. This study confirms crystal‐induced NLRP3 inflammasome activation located at the syncytiotrophoblast layer as a mechanism of placental inflammation and suggests contribution of enhanced NLRP3 activation to the harmful placental inflammation in pre‐eclampsia.

PP042. Cell surface toll-like receptors in primary first trimester trophoblasts

INTRODUCTION The first trimester of pregnancy is characterised by a mild pro-inflammatory environment, however excessive inflammation threatens placental development and function. Toll-like receptors (TLRs) are crucial in initiating inflammation. TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 are expressed on the cell surface, and respond to microbial infection and cell damage and stress signals. Recent findings of TLRs in trophoblasts indicate a role in inflammation during pregnancy, but further studies are warranted. OBJECTIVES To investigate gene expression and function of cell surface TLRs in first trimester trophoblasts, to extend knowledge on the role of trophoblast TLRs during placental development. METHODS Primary trophoblasts were isolated from first trimester placentas (n=6) by enzyme degradation and density gradient centrifugation. Gene expression of TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 was quantified by RT-qPCR in primary first trimester trophoblasts and the trophoblast cell line BeWo. Trophoblasts were stimulated with cell surface TLR ligands and pro-inflammatory cytokine release was analysed by multiplex immunoassay. RESULTS Primary first trimester trophoblasts expressed all cell surface TLR mRNAs, and activation of TLR2/1, TLR4 and TLR5 induced IL-6 and/or IL-8. CONCLUSION The broad expression of functional cell surface TLRs in primary first trimester trophoblasts suggests a central role for trophoblasts in placental inflammation and immune activation.

PP040. Activation of endosomal toll-like receptors in first trimester trophoblasts

INTRODUCTION A mild systemic inflammation may be beneficial to normal pregnancy, however exaggerated inflammation may contribute to pregnancy complications. Infections and cell stress or damage may evoke placental inflammation by activation of Toll-like receptors (TLRs). TLR3, TLR7, TLR8 and TLR9 are located intracellulary on endosomes and are activated by nucleic acids from microbes and damaged cells. Trophoblasts play a crucial role during placentation, and the role of TLRs in first trimester trophoblasts needs to be determined. OBJECTIVES To characterize endosomal TLR gene expression and activation in first trimester trophoblasts, to extend knowledge of endosomal TLR involvement in placental inflammation. METHODS Primary trophoblasts were isolated from six first trimester placentas by enzyme degradation and gradient centrifugation. Gene expression of TLR3, TLR7, TLR8 and TLR9 in primary first trimester trophoblasts and the trophoblast cell line BeWo was quantified by RT-qPCR. The trophoblasts were stimulated with ligands for endosomal TLRs, and release of pro-inflammatory cytokines was analyzed by multiplex. RESULTS Primary first trimester trophoblasts showed gene expression of all endosomal TLRs, and endosomal TLR activation gave increased production of the pro-inflammatory cytokines IL-6, IL-8, and IP-10. CONCLUSION Primary first trimester trophoblasts express functional endosomal TLRs, indicating TLR-mediated trophoblast involvement in early placental inflammation.